Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

Nature Communications
Thapakorn JaroentomeechaiMatthew P DeLisa

Abstract

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

References

May 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·V R LingappaG Blobel
Jun 1, 1991·Molecular and Biochemical Parasitology·S N MorenoG A Cross
Jan 1, 1994·Microbiology·D Liu, P R Reeves
Jul 1, 1996·Neurochemical Research·M ShibutaniG Guroff
Jan 1, 1997·Critical Reviews in Biochemistry and Molecular Biology·P M Rudd, R A Dwek
Dec 22, 1999·Current Opinion in Chemical Biology·B Imperiali, S E O'Connor
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Mar 28, 2001·Science·A Helenius, M Aebi
Mar 28, 2001·Science·P M RuddR A Dwek
Aug 30, 2003·Science·Stephen R HamiltonTillman U Gerngross
Mar 10, 2004·Biotechnology and Bioengineering·Michael C Jewett, James R Swartz
Jul 21, 2004·Journal of Computational Chemistry·Eric F PettersenThomas E Ferrin
Feb 11, 2005·Proceedings of the National Academy of Sciences of the United States of America·Mario F FeldmanMarkus Aebi
Jun 17, 2005·Journal of Pharmaceutical Sciences·Angus M Sinclair, Steve Elliott
Mar 3, 2006·Glycobiology·Eranthie Weerapana, Barbara Imperiali
Aug 8, 2006·Journal of Biotechnology·Satoshi MikamiHiroaki Imataka
Aug 8, 2007·Current Opinion in Chemical Biology·Pascal M LanctotAjit P Varki
Oct 9, 2008·Proceedings of the National Academy of Sciences of the United States of America·Liselotte KaiserShuguang Zhang
Oct 16, 2008·Molecular Systems Biology·Michael C JewettJames R Swartz
Mar 2, 2010·Nature Chemical Biology·Flavio SchwarzLai-Xi Wang
Jun 17, 2011·Nature·Christian LizakKaspar P Locher
Oct 20, 2011·Biotechnology Advances·Erik D CarlsonMichael C Jewett
Nov 10, 2011·Glycobiology·Cassandra Guarino, Matthew P DeLisa
Mar 6, 2012·Journal of Bacteriology·Adrian J JervisDennis Linton
Mar 27, 2012·Nature Chemical Biology·Juan D Valderrama-RinconMatthew P DeLisa
Mar 5, 2013·Biotechnology and Bioengineering·Judith H MerrittMatthew P DeLisa
Nov 16, 2013·Nature Chemical Biology·Margreet A Wolfert, Geert-Jan Boons
Apr 23, 2014·Nature Biotechnology·Leander MeurisNico Callewaert
Jul 11, 2014·Integrative Biology : Quantitative Biosciences From Nano to Macro·Kirsten JacksonZ Hugh Fan
Oct 18, 2014·Nature Chemical Biology·Daniel N HebertJeffery W Kelly
Nov 25, 2014·Cell·Keith PardeeJames J Collins
Dec 3, 2014·Journal of Bioscience and Bioengineering·Akkaraphol SrichaisupakitKazuhito Fujiyama
Dec 17, 2014·Nature Structural & Molecular Biology·Gregor HageluekenJames H Naismith
Mar 3, 2015·Scientific Reports·Yong-Chan Kwon, Michael C Jewett

❮ Previous
Next ❯

Citations

Dec 30, 2018·Biotechnology and Bioengineering·Jared Lynn DoppNigel Forest Reuel
Apr 17, 2019·Glycobiology·Christian M Harding, Mario F Feldman
Jun 6, 2019·Methods and Protocols·Nicole E GregorioJavin P Oza
Jan 3, 2020·Lab on a Chip·Junjie ZhongDavid Sinton
Aug 8, 2018·Science Advances·Jessica C StarkMichael C Jewett
Feb 7, 2020·International Journal of Molecular Sciences·Sviatlana SmolskayaYaroslav A Andreev
Nov 30, 2019·Nature Communications·Weston KightlingerMichael C Jewett
Nov 11, 2019·Nature Communications·Joongoo LeeMichael C Jewett
Mar 11, 2020·Annual Review of Biomedical Engineering·Vincent Noireaux, Allen P Liu
Jul 29, 2020·Nature Chemical Biology·Aravind NatarajanMatthew P DeLisa
Jun 21, 2020·Nature Communications·Luis E Contreras-LlanoCheemeng Tan
Dec 17, 2019·Biotechnology and Bioengineering·Mohammad H Ayoubi-JoshaghaniRana Jahanban-Esfahlan
Mar 22, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Srujan Kumar DondapatiStefan Kubick
Feb 6, 2020·Nucleic Acids Research·Anne E d'AquinoMichael C Jewett
Sep 3, 2019·Expert Review of Vaccines·Francesca MicoliRoberto Adamo
Sep 20, 2020·Science Advances·Ö Duhan ToparlakSheref S Mansy
Nov 5, 2019·Frontiers in Pharmacology·Kyung-Ho Lee, Dong-Myung Kim
Aug 10, 2019·BMC Biology·Aidan TinafarKeith Pardee
Oct 23, 2020·Journal of Industrial Microbiology & Biotechnology·Jasmine HersheweMichael C Jewett
Oct 30, 2020·Frontiers in Bioengineering and Biotechnology·Zhenling CuiKirill Alexandrov
Oct 26, 2018·Emerging Topics in Life Sciences·Aravind NatarajanMatthew P DeLisa
Feb 3, 2021·Nature Communications·Evan AmalfitanoKeith Pardee
Feb 5, 2021·Science Advances·Jessica C StarkMichael C Jewett
Jun 6, 2020·Metabolic Engineering·William S GrubbeAshty S Karim
Mar 18, 2021·Advanced Biology·Nathaniel J Gaut, Katarzyna P Adamala
Jun 12, 2020·ACS Synthetic Biology·Weston KightlingerMichael C Jewett
Aug 11, 2020·Synthetic and Systems Biotechnology·Stephanie D ColeMatthew W Lux
Mar 23, 2021·ACS Synthetic Biology·Nicholas S KruyerPamela Peralta-Yahya
May 14, 2021·Proceedings of the Japan Academy. Series B, Physical and Biological Sciences·Yaeta Endo
Jun 29, 2021·Critical Reviews in Biochemistry and Molecular Biology·Matthew WongMattheos Koffas
Jul 3, 2021·Life·Conary MeyerCheemeng Tan
Jul 1, 2021·Journal of the American Chemical Society·Kota NomuraYasuhiro Kajihara
Aug 15, 2018·ACS Synthetic Biology·Benjamin J Des SoyeMichael C Jewett
Dec 20, 2019·Analytical Chemistry·José-Marc TechnerMilan Mrksich
Sep 16, 2020·Bioconjugate Chemistry·Nicholas HolloranPhilip Garner
Aug 27, 2021·Metabolites·Jeffrey PattersonHaiwei Gu

❮ Previous
Next ❯

Methods Mentioned

BETA
glycosylation
dot blot
PCR
size exclusion chromatography
organic solvent extraction

Software Mentioned

PURExpress
CFGpS
Byonic
Byonics
Xcalibur
Orbitrap Fusion Tune Application

Related Concepts

Related Feeds

Bioinformatics in Biomedicine

Bioinformatics in biomedicine incorporates computer science, biology, chemistry, medicine, mathematics and statistics. Discover the latest research on bioinformatics in biomedicine here.